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ORIGINAL RESEARCHpublished: 04 October 2017

doi: 10.3389/fpls.2017.01729

Edited by:Jacqueline Batley,

University of Western Australia,Australia

Reviewed by:Hussein Shimelis,

University of KwaZulu-Natal,South Africa

David S. Douches,Michigan State University,

United States

*Correspondence:Qin Chen

chenpeter2289@nwsuaf.edu.cnYue Chen

353606197@qq.com

†These authors have contributedequally to this work and should be

considered co-first authors.

Specialty section:This article was submitted to

Crop Science and Horticulture,a section of the journal

Frontiers in Plant Science

Received: 26 July 2017Accepted: 21 September 2017

Published: 04 October 2017

Citation:Yang L, Wang D, Xu Y, Zhao H,

Wang L, Cao X, Chen Y and Chen Q(2017) A New Resistance Gene

against Potato Late Blight Originatingfrom Solanum pinnatisectum Located

on Potato Chromosome 7.Front. Plant Sci. 8:1729.

doi: 10.3389/fpls.2017.01729

A New Resistance Gene againstPotato Late Blight Originating fromSolanum pinnatisectum Located onPotato Chromosome 7Le Yang†, Dongdong Wang†, Yong Xu, Hua Zhao, Lei Wang, Xiaoli Cao, Yue Chen* andQin Chen*

State Key Laboratory of Crop Stress Biology for Arid Areas, College of Agronomy, Northwest A&F University, Yangling, China

Late blight, caused by the pathogen Phytophthora infestans, is one of the mostdevastating diseases of potato. Here, we describe a new single dominant resistancegene, Rpi2, from the Mexican diploid wild species Solanum pinnatisectum that confershigh level and broad spectrum resistance to late blight. The Rpi2 locus confers fullresistance to complex isolates of P. infestans, for which race specificity has not yetbeen demonstrated. This new gene, flanked by the RFLP-derived marker SpT1756 andAFLP-derived marker SpAFLP2 with a minimal genetic distance of 0.8 cM, was mappedto potato chromosome 7. Using the genomic sequence data of potato, we estimatedthat the physical distance of the nearest marker to the resistance gene was about27 kb. The map location and other evidence indicated that this resistance locus wasdifferent from the previously reported resistance locus Rpi1 on the same chromosomefrom S. pinnatisectum. The presence of other reported resistance genes in the targetregion, such as Gro1-4, I-3, and three NBS-LLR like genes, on a homologous tomatogenome segment indicates the Rpi2-related region is a hotspot for resistance genes.Comparative sequence analysis showed that the order of nine markers mapped to theRpi2 genetic map was highly conserved on tomato chromosome 7; however, somerearrangements were observed in the potato genome sequence. Additional markersand potential resistance genes will promote accurate location of the site of Rpi2 andhelp breeders to introduce this resistance gene into different cultivars by marker-aidedselection.

Keywords: potato, late blight, resistance gene, genetic map, collinearity analysis

INTRODUCTION

Late blight caused by Phytophthora infestans is one of the most devastating diseases of potatoworldwide. Under favorable conditions for the pathogen, complete defoliation of a potato plantcan occur in just a few weeks. The development and deployment of cultivars with genetic resistanceis the most economical and eco-friendly approach for reducing yield losses due to late blight.Wild potato species are a valuable genetic pool for finding late blight resistant genes. The firstparadigm came from the hexaploid Mexican wild species Solanum demissum. Eleven resistance(R) genes, named R1 to R11, were identified in this wild species and introduced into S. tuberosum

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(Black, 1951; Black et al., 1953; Malcolmson and Black, 1966).However, these R genes conferred race-specific resistance andthose that were introgressed into potato varieties were quicklyovercome by the pathogen because of its high genetic variability(Wastie, 1991). Hence, new sources of resistance are required,especially those conferring race non-specific resistance to lateblight.

The co-evolution of the pathogen and wild species in CentralAmerica indicated the possibility of finding resistance in speciesfrom Mexico such as S. bulbocastanum, S. pinnatisectum, andS. trifidum. A set of late blight resistance genes has already beenidentified in these species. Notably, in S. bulbocastanum, fourdifferent loci with broad spectrum late blight resistance have beenidentified, namely Rpi-Blb1/RB (Helgeson et al., 1998), Rpi-blb2(van der Vossen et al., 2005), Rpi-blb3 (Park et al., 2005a), andRpi-apbt (Park et al., 2005b). Recently, several other wild Solanumspecies have been reported as potential sources of resistance, suchas S. mochiquense (Jones et al., 2013), S. chacoense (Vossen et al.,2011), and S. × edinense (De Vetten et al., 2014). Sustainablebreeding efforts using these resistance sources have resulted inseveral new potato cultivars (Jo et al., 2014; Haesaert et al., 2015).

Extensive investigations have shown that the molecular basisof R gene resistance is a gene family characterized by twodomains, the nucleotide binding site (NBS) and leucine-richrepeat (LRR) domains (Martin et al., 2003). The conservednature of the motifs within these domains has been exploitedto search for new resistance gene-like sequences or resistancegene analogs (RGAs) using a homology-dependent PCR-basedapproach (Kanazin et al., 1996; Leister et al., 1996; Chen et al.,1998; Hayes and Maroof, 2000). Many RGAs have been mappedto genomic positions containing known resistance specificities,and RGAs have been shown to be derived from knownresistance genes (Collins et al., 1999). Thus, RGAs representcandidates for functional resistance genes. NBS-LRR genes cangenerally be divided into two distinct groups: one encoding anN-terminal domain with Toll/Interleukin-1 Receptor homology(TIR-NBS-LRR) and the other with an N-terminal coiled-coilmotif (CC-NBS-LRR) (Martin et al., 2003). So far, over 20late blight resistance genes, such as R1, R2, R3a, R3b, RB,Rpi-blb2, Rpi-blb3, Rpi-abpt, Rpi-sto1, Rpi-pta1, Rpi-vnt1.1, andRpi-vnt1.3, which all belong to the CC-NBS-LRR class, havebeen cloned (Ballvora et al., 2002; Song et al., 2003; van derVossen et al., 2003; Huang et al., 2005; Vleeshouwers et al.,2008; Foster et al., 2009; Lokossou et al., 2009; Pel et al., 2009).The publishing of the potato genome sequence derived fromthe S. tuberosum Group Phureja clone DM1-3 516 R44 (DM)accelerated the identification of 438 NB-LRR type genes from∼39,000 potato gene models, and will increase the velocity offunctional NB-LRR gene cloning from Solanum species (Jupeet al., 2012).

High level and broad spectrum late blight resistance hasalso been observed in the Mexican diploid wild speciesS. pinnatisectum (Menke et al., 1996; Chen et al., 2003).Compared with S. bulbocastanum, S. pinnatisectum has receivedless attention in late blight research. Kuhl et al. (2001) screened13 accessions of S. pinnatisectum and found that most wereresistant to late blight. Chen et al. (2003) revaluated the late

blight resistance of S. pinnatisectum (PI275233) and found thatit showed broad-spectrum resistance against various knownP. infestans strains including the R9 isolate. They also founddifferent levels of resistance among different accessions ofS. pinnatisectum, suggesting the presence of different resistancegenes. To date, only Kuhl et al. (2001) have reported the geneticanalysis and identification of a single dominant resistance locusin S. pinnatisectum (PI253124), Rpi1, which was mapped tochromosome 7 in an interval of 14.6 cM between two RFLPmarkers, CP56 and TG20A.

The hybridization barrier between S. pinnatisectum andcultivated potatoes had been overcome by the combination ofthe Sli gene and chromosome-doubling techniques (Sanetomoet al., 2014). Therefore, the wild species S. pinnnatisectumshould receive more attention as a resource for potato lateblight resistance breeding. The objective of this study wasto characterize and map a late blight resistance gene fromS. pinnatisectum (PI275233) through genetic linkage analysis andcollinearity analysis. This gene may be useful for developingpotato cultivars with broad spectrum resistance.

MATERIALS AND METHODS

Plant MaterialsA backcross population was developed by crossing the susceptiblediploid S. cardiophyllum (PI186548) as the male parent with theresistant diploid S. pinnatisectum (PI275233). Several clones of asingle resistant F1 individual, propagated through in vitro culture,were then backcrossed with the susceptible parent to generate abackcross mapping population.

The F1 and BC1 populations were maintained vegetativelyfrom tubers following their first propagation from true seed.A total of 931 clones from the backcross population wereselected to analyze the genetics of resistance to late blight usingdetached leaf methods at the first clonal generation (Chen et al.,2003).

Detached Leaf Assay for Evaluating LateBlight ResistanceAn inoculum was prepared from the P1801C.16 strain ofP. infestans (US-8/A2 mating type) and diluted to a finalconcentration of 30,000 sporangia per ml. Inoculation and thedetached leaf assay were performed according to Chen et al.(2003). Three compound leaves were excised for the late blighttest, including the third to fifth leaves from the top on each plant’smain branch. Each compound leaf with five leaflets was insertedinto prepared moist vermiculite in a plastic tray. The inoculumwas sprayed onto the surface of all leaflets. Trays with inoculatedcompound leaves were incubated in a growth cabinet under an18/6 h and 20/18◦C day/night regime for about 15 days. Thesusceptible parent was inoculated as a susceptible check. Plantresistance was evaluated after 8 and 15 days. Disease severitywas estimated using mean disease severity values (DSVs) ofthree compound leaves based on the percentage of leaf area withsymptoms of late blight. Severity values were scored using a scaleof 0–5 where 0 = no disease to <3%; 1 = 3–24%; 2 = 25–49%;

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3 = 50–74%; 4 = 75–94%; and 5 = 95–100% infection. Plantswith a DSV of 0 were classified as resistant and those with DSVsof 2–5 were classified as susceptible.

AFLP AnalysisDNA was extracted from 100 mg of young leaves for eachpotato plant using a Genomic DNA Purification Kit (Promega,Fitchburg, WI, United States). Bulked segregant analysis (BSA)was used to screen for molecular markers associated with lateblight resistance (Michelmore et al., 1991). Two susceptible bulkswere constructed from 10 highly susceptible individuals and aresistant bulk was developed with equal amounts of DNA from10 highly resistant individuals among the BC1 population. DNAmarkers were screened for the two susceptible bulks, the resistantbulk and the resistant parent plant.

AFLP analysis was performed as described by Vos et al.(1995) using EcoRI and MseI as rare- and frequent-cutterenzymes, respectively. Genomic DNA digestion and ligationwere conducted using an AFLP Core Reagent Kit (Invitrogen,Carlsbad, CA, United States) according to the instructions.A pre-amplification was carried out with 1-bp extension primercombinations (EcoRI+A/MseI+C and EcoRI+A/MseI+A) andthe PCR products were diluted at a ratio of 1:30 with TE buffer.Selective amplification using primer combinations of EcoRI+3and MseI+3 was conducted and the products were separatedon a 6% PAGE sequencing gel run at 100 W for 2.5 h afterpre-electrophoresis for 30 min. The gel was stained by the silver-staining method (Bassam et al., 1991).

DNA Sequencing and AnalysisAFLP fragments were excised from the dried silver-stainedpolyacrylamide gel and placed into microfuge tubes containing30 µl distilled water. The samples were boiled for 10 min andcentrifuged, and then 3 µl of the supernatant was used for PCRunder the same conditions as those used for AFLP analysis.The PCR products were then inserted into the pGEM-T easyvector (Promega, Fitchburg, WI, United States) and sequenced.A search for sequences homologous to the AFLP fragments wasconducted using the GenBank website1, and Clustal W22 wasused to compare the sequence homology.

RGA MarkersThe digestion of genome DNA were performed by two restrictionenzymes, EcoRI and MseI, according to the instructions of AFLPCore Reagent Kit. The primer combination EcoRI/MseIwas used to generate pre-amplification products. Then, thesecond amplification step was carried out with five primercombinations. The primer combinations were respectivelycombined by five P-loop based RGAs primers, S1 (5′-GGTGGGGTTGGGAAGACAACG-3′), S2 (5′-GGIGGIGTIGGIAAIACIAC-3′), AS1 (5′-CAACGCTAGTGGCAATCC-3′), AS2(5′-IAAIGCIAGIGGIAAICC-3′) and AS3 (5′-IAGIGCIAGIGGIAGICC-3′) (Leister et al., 1996), with the EcoRI AFLPprimer. PCR conditions were somewhat different from the

1https://www.ncbi.nlm.nih.gov/genbank/2http://www.ebi.ac.uk/Tools/msa/clustalw2/

standard AFLP procedure; 30 s DNA denaturation at 94◦C, 30 sprimer annealing at 55◦C and a 1 min elongation step at 72◦C(35 cycles). Prior to the cycling, the template DNA was denaturedfor 1 min at 94◦C and the PCR was finalized by applying an extra5 min elongation step at 72◦C. The procedures for running thegel and fragment extraction were the same as described for AFLPsection.

Locus-Specific Marker DevelopmentLocus-specific markers on chromosome 7 of potato and tomato3

were selected to develop PCR-based markers. Generally, theRFLP probe sequences were used as queries to search ESTs usingthe BLASTn program (Altschul et al., 1997). Then, the ESTsand RFLP probes were assembled with a criterion of more than95% identity over a stretch of 40 nucleotides using SeqManII (LASERGENE, DNASTAR, Madison, WI, United States).Primers were designed according to the assembled sequencesguided by intron finder4 to amplify regions spanning intronsand avoid placing primers in exon–intron boundaries. ThePCR products were separated after digestion with one of the4-bp cutter restriction enzymes TaqI, Trul1, Msp1, Rsa1, andTail1.

The PCR amplification reactions were conducted in 20 µlreaction mixtures containing 10 mM TRIS-HCl, pH 8.3, 50 mMKCl, 2 mM MgCl2, 100 µM of each dNTP, 200 nM primers,approximately 20 ng template DNA and 1 Unit Taq DNApolymerase. The cycling program consisted of an initial 3 mindenaturation step at 94◦C, followed by 35 cycles of 94◦C (30 s),55◦C (30 s), and 72◦C (50 s), and a final 5 min extension step at72◦C. The PCR products were size-separated on a 2% agarose gel,stained with ethidium bromide, and visualized on a Gel Imagingsystem (Bio-Rad, San Diego, CA, United States).

Linkage AnalysisLinkage analysis was performed using the software packageMAPMAKER V3.0 (Lander et al., 1987). Markers and theircorresponding distances (cM) were included in the frameworkmap only if the LOD value for the ripple was >3. The Kosambimapping function was employed to convert recombinationfrequencies to map distances in cM (Kosambi, 1943). Collinearityanalysis results were visualized using Circos-0.67 (Krzywinskiet al., 2009).

RESULTS

The BC1 generation produced 440 resistant plants and 491susceptible plants. The segregation ratio fit a monogenicMendelian inheritance model of 1:1 (resistant:susceptible) in thepopulation (χ2

= 2.794, P = 0.095). This result suggested thata single dominant locus controlled the late blight resistance inS. pinnatisectum (PI275233). Subsequently, 164 susceptible and101 resistant plants with extreme phenotypes were chosen formapping.

3https://solgenomics.net/cview/index.pl4http://ftp.sgn.cornell.edu/tools/intron_detection/find_introns.pl

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AFLP and AFLP-Derived MarkersIn an attempt to find AFLP markers linked to the resistancelocus, 324 EcoRI+3/MseI+3 (196 E-A/M-C and 128 E-A/M-A)AFLP primer combinations were screened in the bulk materialusing a BSA strategy. Ten putative AFLP fragments wereidentified and segregation analysis in the BC1 populationconfirmed that seven of them were associated with theresistance locus. The two closest markers, EAGCMCGA-450and EACAMAGG-330, were determined to be linkedto the resistance locus at distances of 1.2 and 0.8 cM,respectively.

These two AFLP fragments were cloned and sequenced.BLAST analysis showed that the sequence of EAACMATC-330had no similarity to any known sequence in GenBank, whereasEAGCMCGA-450 hit four potato ESTs (BQ509088, BG600948,DV623421, DV623416). These four ESTs and two other potatoESTs were assembled into a 1720-bp contig with a completecoding region that showed high similarity to the Arabidopsisgene GLUCAN SYNTHASE-like 7 (1e-139) in Blastx5) analysis.Based on this assembled sequence, a CAPS marker ofEAGCMCGA-450, SpAFLP1, was developed (Figures 1A,D). Inaddition, EAACMATC-330 was converted into the CAPS markerSpAFLP2 (Figures 1B,E).

Integration of Rpi2 into the SGN MapThe 1720-bp contig of EAGCMCGA-450 was used to searchthe high-throughput genomic sequence (HTGS) database withBLASTn6 and a tomato BAC (C07HBa0116M01) was identified(e-112). Annotation of this BAC (C07HBa0116M01) revealed apartial VPS16-like gene in the 3′ terminus. This partial genesequence was used as a query to search the EST database withBLASTn and 23 matching ESTs were identified. All 23 ESTs wereassembled to a 2.4-kb contig, named cEST1. BLAST analysisshowed that this sequence had homology to an RFLP marker,TG572 (e-120), which was mapped to tomato chromosome 7.Subsequently, a CAPS maker named SpTG572 was developedaccording to this sequence and was shown to co-segregate withSpAFLP1 (Figure 2).

The 5′ terminal sequence of BAC C07HBa0116M01 was usedto build a 2-kb contig with 10 ESTs, named cEST2. This contighit a potato BAC end sequence, POTDQ81TR. Using the otherend sequence POTDQ81TF as a query, we identified a tomatoBAC, C07HBa0018L21. A PCR marker, SpAL21, was developedbased on the left end sequence of BAC C07HBa0018L21 andrecombination was found between this marker and SpTG572(Figure 2).

TG572 was near I-3, a gene for fusarium wilt resistance fromthe wild tomato species Lycopersicon pennellii, with a geneticdistance less than 0.3 cM (Hemming et al., 2004). Two additionalmarkers closely linked to I-3, CT226, and Got2, were converted toSCAR markers in our mapping population, and named SpCT226and SpGot2, respectively. Segregation analysis indicated SpCT226

5https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastx&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome6https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome

and SpGot2 were proximal and distal with genetic distances of 2.8and 3.2 cM, respectively (Figure 3B).

The flanking markers TG572, T0810, and T1756 in the SGNmap7 were developed into PCR-based markers and tested inthe mapping population. The results delimited the resistancelocus to the interval between StAFLP2 and SpT1756 on potatochromosome 7 (Figure 3B).

An RGA Flanks the Resistance LocusThe RGA fingerprinting technique was used to identifyfunctionally relevant markers linked to the resistance for lateblight. An RGA fragment, RGA1, amplified by the AS2 primer(Leister et al., 1996) in combination with the E00 AFLP primer,did not exist in the two susceptible bulks but appeared in theresistant bulk and the resistant parent (Figure 1C). Sequenceanalysis of this 320-bp long fragment revealed homology to anRGA sequence previously mapped to the long arm of potatochromosome 7, Gro1-5 (Leister et al., 1996). Therefore, thisfragment was named SpGrol-1. A PCR marker was developed andsegregation analysis indicated that SpGrol-1 was 2.8 cM from theresistance locus (Figure 1F).

Genetic Relationship between Rpi1 andRpi2Previously, the late blight resistant locus Rpi1, also derived fromS. pinnatisectum, was assigned to chromosome 7 flanked bytwo RFLP markers, TG20 and CP56 (Kuhl et al., 2001). Tocompare the map positions of Rpi1 and our target resistancelocus, the RFLP markers TG20, CP56 and their interval markerGP1278 were converted into PCR-based markers. The markerinformation is listed in Supplementary Table S1. Segregationanalysis of these converted PCR-based markers showed a linkbetween the late blight resistance loci. The genetic distancebetween CP56 and TG20 was 15.1 cM, similar to the map ofKuhl et al. (2001) (14.6 cM). However, there was an obviousdifference in the genetic distance between the markers linked tothe resistance genes. CP56 and TG20 were 4.0 and 11.1 cM awayfrom our target gene, respectively, which was different to thegenetic distances of Rpi1 to these two markers (9.4 and 5.2 cM,respectively) (Figures 3A,B). Therefore, our resistance gene wascalled Rpi2.

Although the two resistance genes were derived from thesame species, they came from two different accessions; Rpi1 fromPI235214 and Rpi2 from PI 275233. To further identify geneticdifferences between the accessions PI275233 and PI235214, wescreened all of the markers and found that the two accessions haddifferent genotypes at two loci, SpAFLP2 and SpCT226.

Collinearity Analysis of TargetChromosome Regions between Potatoand TomatoThe molecular marker sequences were used as queries tosearch for homologous loci in the genome sequence databases

7https://solgenomics.net/cview/index.pl8https://solgenomics.net/marker/SGN-M16437/details

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FIGURE 1 | AFLP and RGA markers and their conversion into PCR-based markers. (A) Marker EAGC/MCGA-450 and (D) its SCAR marker SpAFLP1. (B) MarkerEAAC/MATC-330 and (E) its SCAR marker SpAFLP2. (C) The RGA1 marker and (F) its SCAR marker SpGrol-1. Bs1, Susceptible bulk 1; Bs2, susceptible bulk 2;Br, resistant bulk; Ps, susceptible parent; Pr, resistant parent.

FIGURE 2 | A contig built near the Rpi2 locus. Three BACs (C07HBa0018L21, POTDQ81, and C07HBa0116M01) and two EST assembles (cEST1 and cEST2)overlapped and were assembled into a contig. Two markers located at either end of the contig were developed, SpAL21 and SpTG572. Ps, Susceptible parent; Pr,resistant parent.

FIGURE 3 | Genetic maps of the late blight resistance gene Rpi2 and their correspondence to comparative genomic maps of DM and tomato. (A) A genetic map ofRpi1 established by Kuhl et al. (2001). (B) A genetic map of Rpi2 established in this study. (C) Homologous markers on Solanum tuberosum group PhurejaDM1-3(DM) chromosome 7. (D) Annotated genes on DM chromosome 7 between StAFLP1 and StAL21. (E) Annotated genes on Solanum lycopersicum (SL2.50)chromosome 7 between SlAFLP1 and SlAL21. (F) Homologous markers on S. lycopersicum chromosome 7. Collinear loci are indicated by black lines.

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FIGURE 4 | Collinearity analysis of target chromosome regions between potato and tomato. (A) The chromosome region between SlT1756 and SlTG572, whichcontains 10 annotated genes in the tomato genome. (B) Collinearity analysis of Rpi2-related chromosome regions between potato (DM v4.03) and tomato (SL2.50).(C) A 50-kb gap between StT1756 and StAFLP2 in the potato genome.

of potato and tomato. The majority of markers linked toRpi2 showed homology to STChr7 and SLChr7, and wereincluded in two orthologous genomic regions spanning 2.9and 2.4 Mb, respectively (Figure 4B). Specifically, 9 of the 11molecular markers mapped in the Rpi2 genetic linkage mapgenerated hits to 16 homologous loci in STChr7 and 10 loci inSLChr7 (Figures 3C,F). Because cloning and sequencing failed,homologous loci of SpGot2-1 and SpTG20 were not found.

Comparative genomic analysis revealed that 14 of the 17annotated genes in potato between StAFLP1 and StAL21 hadsimilarity to corresponding regions in tomato (Figures 3D,E),again revealing high levels of collinearity in the Rpi2 regionbetween potato and tomato. Furthermore, the order of thesesimilar genes was highly conserved but reversed. Segmentalinversion, which was a reasonable explanation for the reverseorder of the conserved genes, was observed on STChr7 betweenStAFLP1 and StAL21 compared with the Rpi2 linkage map andSLChr7.

Scanning the Spud DB Genome Browser for Potato (Solanumtuberosum group Phureja DM1-3) PGSC v4.03 Pseudomolecules9

suggested the physical distance between StAFLP2 and StT1756was about 84 kbp. Consequently, we estimated that the physicaldistance between Rpi2 and SpAFLP2 was about 28 kbp byreferring to their genetic distance. Unfortunately, there wasa 50-kbp gap in this potato genome region that potentiallycontained the homologous gene of Rpi2 (Figure 4C). However,the homologous segment in the tomato genome was assembledcompletely, in which 3 of 10 loci Table 1 were annotatedas NBS-LRR class disease resistance proteins (Accession nos.Solyc07g056180.1, Solyc07g056190.2, and Solyc07g056200.2)(Figure 4A).

9http://solanaceae.plantbiology.msu.edu/cgi-bin/gbrowse/potato/

DISCUSSION

The short-lived R genes from S. demissum prompted potatobreeders and geneticists to look for resistance genes in other wildSolanum species (Van Soest et al., 1984; Colon and Budding,1988; Douches et al., 2001). High-level resistance has been foundin several diploid Mexican species, including S. bulbocastanumand S. pinnatisectum (Helgeson et al., 1998; Kuhl et al., 2001;Chen et al., 2003). These species may have adapted to coexistwith highly complex and dynamic P. infestans populations(Niederhauser, 1953; Niederhauser et al., 1954). Genetic mappingstudies indicated that the resistance in both S. bulbocastanumand S. pinnatisectum might be conferred by a single gene ora few dominant genes (Naess et al., 2000; Kuhl et al., 2001).Here, we identified a single dominant late blight resistancegene from the wild potato species S. pinnatisectum (PI 275233)and mapped it to an interval of 2.4 cM on the long arm ofchromosome 7.

A Hotspot of Resistance Genes onPotato Chromosome 7Accumulated evidence has suggested that resistance loci arenot distributed randomly along chromosomes. Several hotspotsfor resistance genes have been described in Solanum species.For instance, at least five R genes against diverse pathogenshave been mapped to the GP21–GP179 interval on chromosome5 in different genetic backgrounds; Gpa and Grp1 conferringresistance to potato cyst nematodes (Kreike et al., 1994; vander Voort et al., 1998), Nb and Rx2 conferring resistance topotato virus X (Ritter et al., 1991; DeJong et al., 1997), andR1 conferring resistance to P. infestans (Leonards-Schipperset al., 1992). In the current study, we found that Rpi2 was

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TABLE 1 | Annotated genes in the tomato genome region between SlT1756 and SlTG572.

Name Location on SL2.50ch07 Description InterPro domain

Solyc07g056130.1 63988594..63988983 Unknown protein –

Solyc07g056140.2 63989209..63993821 Glucose-1-phosphate adenylyltransferase IPR011831

Solyc07g056150.2 63994885..63999176 Ras-related protein Rab-2-A IPR003579

Solyc07g056160.2 64018080..64022506 Cytochrome P450 –

Solyc07g056170.2 64023009..64028326 Subtilisin-like protease IPR015500

Solyc07g056180.1 64036615..64037860 NBS-LRR class disease resistance protein –

Solyc07g056190.2 64043548..64044315 NBS-LRR class disease resistance protein –

Solyc07g056200.2 64047159..64048119 NBS-LRR class disease resistance protein –

Solyc07g056210.2 64055352..64056480 Unknown protein –

Solyc07g056220.2 64063658..64074684 Vacuolar sorting-associated protein IPR016534

located in a major cluster on the long arm of chromosome 7in which several R genes have been mapped, including Rpi1conferring resistance to P. infestans, Gro1-4 conferring resistanceto Globodera rostochiensis, and I-3 conferring resistance toFusarium oxysporum (Bournival et al., 1989; Ballvora et al.,1995; Kuhl et al., 2001; Paal et al., 2004; Ruggieri et al., 2014;Catanzariti et al., 2015). Clearly, this region is another hotspot forresistance genes, and can be expected to contain more resistancegenes.

Resistance loci regions are usually enriched in NBS-LRRhomologs. For instance, there are at least 13 TIR-NBS-LRRsequences clustered across more than 400 kb in the locusGro1 (Paal et al., 2004) and one of them, Gro1-4, has beenshown to be responsible for a resistance trait. In this research,we used the RGA profiling strategy to identify an RGAfragment, SpGrol-1, linked to the resistance locus. Sequenceanalysis showed that SpGrol-1 belonged to the TIR-NBS-LRRfamily, and that the most similar sequence was Gro1-5, agene at the Gro1 locus. However, mapping analysis showedthis RGA was proximal to Rpi2 with a genetic distance of2.8 cM.

Resistance gene analogs are generally clustered in the genome(Meyers et al., 1998; Michelmore and Meyers, 1998). Clustersof R genes can be tightly organized or spaced over severalmegabases of sequence (Meyers et al., 1998; Noël et al., 1999).We thought that similar Gro1-like sequences might be presentin our resistance locus. Hence, we designed a set of primersaccording to an alignment of 13 Gro1 sequences and developedthree PCR markers. However, all of these markers co-localized toSpGrol-1 (data not shown). This indicated that there was morethan one Gro1-like sequence and that the Rpi2 gene might not bea Gro1-like gene.

A similar observation was also described for the resistancegene I-3 from the wild tomato L. pennellii (Hemming et al.,2004). I-3 co-segregated with RGA St332; however, RGA St332was ruled out as a candidate gene for I-3 because it wasa single-copy pseudo gene in L. pennellii. I-3 was flankedby two RFLP markers TG572 and CT226 in an interval of0.6 cM. That Rpi2 and I-3 share the flanking markers CT226and TG572 supports that these two genes are in a syntenicregion.

Comparative Sequence Analysis of theRpi2 RegionComparative genomics between potato and tomato facilitatedthe mapping and isolation of the late-blight R genes R3a andRpi-blb2 from potato in a previous study, as these genes weremapped to regions of the potato genome that were syntenic topreviously cloned gene loci (I2 and Mi, respectively) in tomato(Huang et al., 2005; van der Vossen et al., 2005). Recently, boththe potato and tomato genomes have been sequenced (Xu et al.,2011; Sato et al., 2012). This sequence information should greatlyaccelerate the cloning of the Rpi2 gene through comparativegenomics.

Comparing the homologous regions in the potato andtomato genomes, the genetic linkage map of Rpi2 showed highuniformity except that a chromosome inversion had occurred inthe sequenced DM genome (Figure 3). Although this inversionmay be a result of chromosomal variation during evolution,incorrect sequencing or assembly could equally have led to theobserved recombination because short reads, a large amountof repetitive sequence, the sequence GC composition and othereffects can impede uniform and complete sequencing coveragealong the genome (Maiti and Bouvagnet, 2001; van Hijum et al.,2005; Aird et al., 2011; Schatz et al., 2012; Berlin et al., 2015).The gap between SpAFLP2 and SpT1756 on StChr7 indicatedthe accuracy of the assembly in the Rpi2-related region wasnot sufficiently high. In other words, the fragment inversionobserved by comparative analysis was not sufficient evidence todemonstrate chromosome inversion. Furthermore, the lack ofsequence information between the flanking markers preventedus from obtaining candidate genes from the DM genome data.Therefore, constructing a higher quality genome assembly for theRpi2-related region requires enhanced approaches.

At present, an effort to introgress disease resistance genesfrom S. pinnatisectum into potato is being carried outto develop resistant cultivars. Because of the ploidy levelbarrier and endosperm balance number incompatibility, it isdifficult to transfer resistance traits from S. pinnatisectum tocultivated potato. Fortunately, the hybridization barrier betweenS. pinnatisectum and cultivated potatoes can be overcome byembryo rescue, protoplast fusion, and chromosome-doublingtechniques (Chen et al., 2008; Sanetomo et al., 2014). Our

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molecular markers could help breeders to introduce thisresistance gene into different cultivars by marker-assistedselection.

AUTHOR CONTRIBUTIONS

LY and DW: Conducted the experiments, analyzed the data,and wrote the manuscript. YX: Identified the resistant parentalline and made the cross. LW and XC: Participated in detectingmolecular makers and contributed to the genotyping experiment.HZ: Assisted in analyzing the data. YC and QC: Conceived anddirected the project and revised the manuscript.

FUNDING

This study was financially supported by Key Research andDevelopment Program of Shaanxi Province, China (grant no.

2017ZDXM-NY-004) and the Research Funding for SpecialProfessor of Northwest Agriculture and Forestry University(grant no. Z111021202).

ACKNOWLEDGMENTS

We thank Robbie Lewis, M.Sc., from Liwen Bianji, Edanz GroupChina (www.liwenbianji.cn/ac), for editing the English text of adraft of this manuscript.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found onlineat: http://journal.frontiersin.org/article/10.3389/fpls.2017.01729/full#supplementary-material

TABLE S1 | Information of molecular markers involved in this study.

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Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

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